Xenotransplantation with pig organs offers the best near term hope for satisfying the limitation imposed by shortage of human organs. This research project is directed toward producing as a product a genetically modified pig that will be the best potential donor for xenotransplants to humans. Although hyperacute rejection of pig organs in primate recipients has been overcome by the production of (-1,3-galactosyltransferase knockout (GalT-KO) pigs, experiments to date with GalT-KO organs strongly suggest that clinically relevant chronic immunosuppression of recipients will be insufficient to overcome rejection of xenotransplants. Tolerance induction through mixed hematopoietic chimerism is currently the best means for inducing such tolerance, and this methodology has recently been shown to allow human allograft acceptance without chronic immunosuppression. The major problem in achieving mixed chimerism across the pig-to- primate barrier has the difficulty in achieving durable chimerism. Our proposed product will consist of genetically modified pigs with enhanced xenogeneic hematopoietic chimerism potential that we will make available commercially to the research community, thereby providing a strategy for advancing solid organ xenotransplantation toward the clinic. We will achieve this goal by overcoming the species incompatibility of the CD47 (IAP, integrin associated protein) cell surface molecule, which appears to be the key barrier to the establishment of chimerism following pig-to-primate bone marrow transplantation. Ubiquitously expressed, CD47 binds SIRP( receptors on macrophages and thereby inhibits phagocytosis. Expression of primate CD47 on pig cells greatly reduces their susceptibility to phagocytosis by human macrophages. In Phase I of this STTR proposal we developed and verified the tools necessary for production, via nuclear transfer, of pigs expressing primate CD47 on a GalT-KO background. We successfully developed targeting/expression vectors for CD47 and molecular assays for identifying targeted cell clones. We have also verified a primary cell clone isolation and have isolated primary fetal fibroblast lines for use in the production of CD47 transgenic pigs. In Phase II, we now intend to use these tools to produce GalT-KO pigs expressing primate CD47 via nuclear transfer, analyze CD47 expression in these pigs, evaluate the efficacy of CD47 expression using in vitro systems, and perform proof-of-concept transplant experiments to validate the usefulness of this product in achieving mixed xenogeneic chimerism and tolerance. PUBLIC HEALTH RELEVANCE: The demand for transplantable human organs far exceeds the current supply and the gap between demand and supply continues to grow. The transplantation of pig hearts, kidneys and other organs (known as xenotransplantation) offers the opportunity to greatly reduce or eliminate this shortage. Having now established the necessary tools to produce genetically modified pigs that will facilitate such pig-to-human organ transplants, we now intend to produce these pigs as a commercial product that will further the development of the filed of xenotransplantation.